采用流式细胞仪检测被带有荧光报告基因的曲霉（FLARE）分生孢子感染过的小鼠肺部和支气管肺泡灌洗液细胞

Anupam Jhingran; Shinji Kasahara; Tobias M Hohl

doi:10.21769/BioProtoc.1927

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Peer-reviewed

Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia

采用流式细胞仪检测被带有荧光报告基因的曲霉（FLARE）分生孢子感染过的小鼠肺部和支气管肺泡灌洗液细胞

Anupam Jhingran

SK Shinji Kasahara

TH Tobias M Hohl email

发布: 2016年09月20日第6卷第18期 DOI: 10.21769/BioProtoc.1927 浏览次数: 16957

评审: Shanie Saghafian-HedengrenAnonymous reviewer(s)

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Cover of PLOS Pathogens, featuring study using the protocol.

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Abstract

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.

Keywords: Fungus (真菌)

Materials and Reagents

Cell strainers - 40 μm and 100 μm
Syringes
Injector (Modified precision dispensing tips) (Nordson, catalog number: 7018166 )
15 ml tubes
96-well plate, U-bottom
Mice: C57BL/6 (Jackson Laboratories, catalog number: 000664 )
Fungal strains (Af293 and Af293-DsRed) (Jhingran et al., 2012)
FATAL-plus solution (Vortech Pharmaceuticals Ltd, National Drug Code: 0298-9373-68 )
BD BBL^TM Sabouraud dextrose agar (Emmons), slants (pH 6.9) (100/sp) (BD, catalog number: 221827 )
PBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-144 )
Tween 20
Tris base
NaHCO₃
Na₂CO₃
Biotin-XX, SSE (6-((6-((biotinoyl)amino)hexanoyl)amino) hexanoic acid, sulfosuccinimidyl ester, sodium salt) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: B6352 )
DMSO
Streptavidin, Alexa Fluor^® 633 conjugate (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: S-21375 )
Isothesia^TM, Isoflurane (Henry Schein Animal Health, SKU number: 029405 )
RBC lysis buffer (Biolegend, catalog number: 420301 )
DNase I (Roche Diagnostics, catalog number: 10104159001 )
Collagenase type IV (Worthington Biochemical, catalog number: LS004189 )
EDTA
Mouse BD Fc Block^TM (BD, BD Pharmingen^TM, catalog number: 553142 )
Antibodies (listed in Procedure section)
PBS Tween (0.025%) (see Recipes)
Tris-chloride (1 M, pH 8) (see Recipes)
Sodium bicarbonate buffer (0.1 M, pH 9.5) (see Recipes)
Lung digestion buffer (for each lung) (see Recipes)
FACS buffer (see Recipes)

Equipment

Hemocytometer
Microscope
Centrifuge
Plexiglass stand
Dissection scissors and forceps
Bronchoalveolar lavage fluid (BALF) catheters (Exel International, catalog number: 26738 )
4-way large bore (lipid resistant) stopcock with rotating male luer lock adapter (Baxter, catalog number: 2C6204 )
gentleMACS tissue dissociator, MACSMix tube rotator and C tubes (Miltenyi Biotech)
Automated cell counter or hemocytometer
Flow cytometer capable of analyzing at least 7 fluorescent parameters (such as BD LSRII). The 532 or 561 nm (but not 488 nm) laser excites DsRed fluorescence and the 633 nm laser excites Alexa Fluor 633 fluorescence.

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Jhingran, A., Kasahara, S. and Hohl, T. M. (2016). Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia . Bio-protocol 6(18): e1927. DOI: 10.21769/BioProtoc.1927.
Heung, L. J., Jhingran, A. and Hohl, T. M. (2015). Deploying FLAREs to visualize functional outcomes of host-pathogen encounters. PLoS Pathog 11(7): e1004912.